Enhancement of the nutritional or health benefits of oils through genetic engineering is being addressed throughout the agricultural community. Several approaches involve manipulation of already present cellular biosynthetic pathways. Steroid biosynthetic pathways are of current interest, particularly for the enhancement of health benefits from food oils.
Several related U.S. patents address increasing sterol accumulation in higher plants. Those patents include U.S. Pat. No. 5,589,619 “Process and Composition for increasing squalene and sterol accumulation in higher plants” (accumulation of squalene in transgenic plants by increasing HMGR activity) and U.S. Pat. No. 5,306,862 “Method and composition for increasing sterol accumulation in higher plants” (increasing HMGR activity to increase plant sterol accumulation—including sterol and cycloartenol, which affects insect resistance—in tobacco, tomato, corn, carrot, soybean, cotton, barley, arabidopsis, guayule and petunia; seeds with elevated sterol/cycloartenol, 7S promoter and CaMV promoters), U.S. Pat. No. 5,365,017 “Method and composition for increasing sterol accumulation in higher plants” (DNA construct with HMGR-CaMV 35S, transgenic plants, hybrid plants, corn, soy, barley, tomato, Arabidopsis), U.S. Pat. No. 5,349,126 “Process and composition for increasing squalene and sterol accumulation in higher plants” (increase in squalene and sterol accumulation by increasing HMGR activity in transgenic tobacco, cotton, soybean, tomato, alfalfa, Arabidopsis, corn, barley, carrot and guayule plants), and EP 486290 (enhancement of squalene and specific sterol.[squalene zymosterol, cholest-7,24-dienol, cholest-5,7,24-trienol] accumulation in yeast by increasing HMGR activity in yeast deficient in enzymes that convert squalene to ergosterol).
In those patents, the amount of a protein exhibiting 3-hydroxy-3-methylglutaryl Coenzyme-A reductase (HMGR) activity is typically increased. HMGR widens a “bottleneck” near the beginning of a biosynthetic path to steroid production, permitting a higher carbon flux through steroid biosynthetic pathways and resulting in increased sterol accumulation.
U.S. Pat. No. 5,480,805 “Composition for modulating sterols in yeast” (enhancement of delta 8-7 isomerase activity-ERG2 enhances accumulation of specific sterols in yeast).
U.S. Pat. No. 5,460,949 “Method and composition for increasing the accumulation of squalene and specific sterols in yeast” (increasing squalene, zymosterol and specific sterols in yeast by increasing HMGR in yeast having decreased erg5 and erg6 activity—Sc and hamster HMGR).
WO 9845457 (SMTI, Erg6 from A.t., corn, yeast; transgenic plants with altered sterol levels_using DNA encoding an enzyme binding a first sterol and producing a second sterol—altered carotenoid, tocopherol, modified FA levels—HMGR, 5α-reductase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, isopentenyl diphosphate isomerase).
Acetate is the metabolic precursor of a vast array of compounds vital for cell and organism viability. Acetyl coenzyme A (CoA) reacts with acetoacetyl CoA to form 3-hydroxy-3 methylglutaryl CoA (HMG-CoA). HMG-CoA is reduced to mevalonate in an irreversible reaction catalyzed by the enzyme HMG-CoA reductase. Mevalonate is phosphorylated and decarboxylated to isopentenyl-pyrophosphate (IPP). Through the sequential steps of isomerization, condensation and dehydrogenation, IPP is converted to geranyl pyrophosphate (GPP). GPP combines with IPP to form farnesyl pyrophosphate (FPP), two molecules of which are reductively condensed to form squalene, a 30-carbon precursor of sterols.
A key enzyme in sterol biosynthesis is 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-COA reductase or HMGR). Schaller et al. (Plant Physiol. 109: 761-770, 1995) found that over-expression of rubber HMGR (hmg1) genomic DNA in tobacco leads to the overproduction of sterol end-products (sitosterol, campesterol and stigmasterol) up to 6-fold in leaves. Further, the excess sterol was stored as steryl-esters in lipid bodies. HMGR activity was increased by 4- to 8-fold.
Sterols are derivatives of a fused, reduced ring system, cyclopenta-[a]-phenanthrene, comprising three fused cyclohexane rings (A, B, and C) in a phenanthrene arrangement, and a terminal cyclopentane ring (D) having the formula (I) and carbon atom position numbering shown below:
where R is an 8 to 10 carbon-atom side chain.
In plants, squalene is converted to squalene epoxide, which is then cyclized to form cycloartenol (4,4,14α-trimethyl-9β,19-cyclo-5α-cholest-24-en-3β-ol). Cycloartenol has two methyl groups at position 4, a methyl group at position 14, a methylene bridge between the carbon atoms at positions 9 and 19 that forms a disubstituted cyclopropyl group at those positions, and includes an 8-carbon sidechain of the formula: CH3CH(CH2)2CH═C(CH3)2. Squalene epoxide can alternatively be converted into pentacyclic sterols, containing five instead of four rings. Exemplary pentacyclic sterols include the phytoalexins and saponins.
Being one of the first sterols in the higher plant biosynthetic pathway, cycloartenol serves as a precursor for the production of numerous other sterols. In normal plants, cycloartenol is converted to predominantly 24-methylene cycloartenol (4,4,14α-dimethyl-9β,19-cyclo-22,23-dihydro-ergosta-24(28)-en-3-β-ol), cycloeucalenol, (4,14α-trimethyl-9β,19 cyclo-5α-ergosta-24(28)-en-3β-ol), isofucosterol (5α-stigmasta-5-24(28)-dien-3β-ol), sitosterol (5α-stigmasta-5-en-3β-ol), stigmasterol-(stigmasta-5,-22-dien-3β-ol), campesterol (5α-ergosta-5-en-3β-ol), and cholesterol (5α-cholesta-5-en-3β-ol). These transformations are illustrated in FIG. 1.
Although sterols produced by plants, and particularly higher (vascular) plants, can be grouped by the presence or absence of one or more of several functionalities, plant sterols are classified into two general groups herein; i.e., those containing a double bond between the carbon atoms at positions 5 and 6 (delta-5 or Δ5 sterols) and those not containing a double bond between the carbon atoms at positions 5 and 6 (non-delta-5 sterols).
Exemplary naturally-occurring delta-5 plant sterols are isofucosterol, sitosterol, stigmasterol, campesterol, cholesterol, and dihydrobrassicasterol. Exemplary naturally occurring non-delta-5 plant sterols are cycloartenol, 24-methylene cycloartenol, cycloeucalenol, and obtusifoliol. The most abundant sterols of vascular plants are campesterol, sitosterol, and stigmasterol, all of which contain a double bond between the carbon atoms at positions 5 and 6 are classified as delta-5 sterols.
The HMG-CoA reductase enzymes of animals and yeasts are integral membrane glycoproteins of the endoplasmic reticulum. The intact enzyme comprises three regions: a catalytic region containing the active site of the enzyme; a membrane binding region anchoring the enzyme to the endoplasmic reticulum; and a linker region joining the catalytic and membrane binding regions of the enzymes. The membrane binding region occupies the amino-terminal (N-terminal) portion of the intact protein, whereas the catalytic region occupies the carboxy-terminal (C-terminal) portion of the protein, with the linker region constituting the remaining portion. M. E. Basson et al., Mol. Cell Biol., 8(9):3797-3808 (1988).
The activity of HMG-CoA reductase in animals and yeasts is known to be subject to feedback inhibition by sterols. Such feedback inhibition requires the presence of the membrane binding region of the enzyme. See, e.g., G. Gil et al, Cell, 41:249-258 (1985); M. Bard and J. F. Downing, J. Gen. Microbiol., 124:415-420 (1981).
Given that mevalonate is the precursor for sterols and other isoprenoids, it might be expected that increases in the amount or activity of HMG-COA reductase would lead to increases in the accumulation of both sterols and other isoprenoids.
In mutant strains of the yeast Saccharomyces cerevisiae (S. cerevisiae) having abnormally high levels of HMG-COA reductase activity, the production of two sterols, 4,14-dimethylzymosterol and 14-methylfucosterol is markedly increased above normal. Downing, et al., Biochem. Biophys. Res. Comm., 94(3): 874-979 (1980).
When HMG-COA reductase activity was increased by illumination in non-photosynthetic microorganisms, isoprenoid (carotenoid), but not sterol (ergosterol), synthesis was enhanced. Tada, et al., Plant and Cell Physiology, 23(4):615-621 (1982).
WO 9703202 discloses a method for identifying agents modulating sterol biosynthesis using a yeast acetoacetyl CoA thiolase (ERG10) gene linked to a reporter system to evaluate compounds, such as lovastatin and other HMG-CoA synthase inhibitors, that affect cholesterol biosynthesis.
U.S. Pat. No. 5,668,001 teaches a recombinant avian HMG-COA synthase preparation useful for evaluating drugs that inhibit cholesterol biosythesis.
JP 09121863 discloses a plant with increased 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity as a result of increasing the expression of a mutant protein kinase gene that regulates expression of the HMGR gene. The increased HMGR activity increases squalene, zymosterol, cholesta-7,24-dienol and cholest-5,7,24-trenol accumulation in yeast with ERG5 and ERG6 mutants.
EP 480730 “Plant-sterol accumulation and pest resistance-by increasing copy number of 3-hydroxy-3-methyl glutaryl coenzyme-A reductase gene in tobacco, tomato and corn
WO 9913086 “Human Delta 7-sterol reductase polypeptide-useful for diagnosis or treatment of genetic defects e.g. hereditary Smith-Lemli-Opitz syndrome” teaches making and using the recombinant polypeptide with humans.
Chappell et al. U.S. Pat. No. 5,589,619 teaches that transformation of higher plants with truncated HMG-CoA reductase enhanced the production of squalene, cycloartenol and certain sterols, particularly compounds having unsaturations at the 5-position. Several intermediate sterols as are shown in FIG. 1 were also produced. It would be beneficial if the production of sitosterol and stigmasterol could be enhanced while lessening the accumulation of the intermediate sterols. The present invention provides avenues for enhancing production of sitosterol and stigmasterol and lessening the accumulation of the intermediate sterols.
Gonzalez et al. (Abstract of poster at Third Terpnet Meeting of the European Network on Plant Isoprenoids, May 29-30, 1997, Poitiers, France) over-expressed the Arabidopsis HMGR CDNA (hmg1 and hmg2) and found sterol overproduction with hmg1 only. They used two forms of the hmg1 gene, a full-length form and a truncated form containing only the catalytic domain. HMGRs have three domains, an N-terminal membrane spanning domain, a short linker domain, and a C-terminal catalytic domain. In this case the transgenic plants were also Arabidopsis. The difference between the full-length and truncated forms was a greater accumulation of pathway intermediates in the case of the truncated form. More importantly, the intermediates demonstrated as accumulating were cycloartenol, 24-methylenecycloartanol and obtusifoliol.
Finally, U.S. Pat. Nos. 5,365,017 and 5,306,862, both assigned to Amoco Corp., disclose a method for increasing sterol accumulation in plants by increasing the copy number of a gene having HMG-CoA reductase activity. These inventions disclose a method using hamster truncated HMGR that consisted of the catalytic domain and the linker domain. According to the claims the linker domain was essential for activity. They also demonstrated a greater accumulation of pathway intermediates such as cycloartenol.